Solid-State NMR Structural Studies of Proteins Using Paramagnetic Probes

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1 Solid-State NMR Structural Studies of Proteins Using Paramagnetic Probes Christopher Jaroniec Department of Chemistry & Biochemistry The Ohio State University

2 Protein Structure by MAS Solid-State NMR D γγ / r 3 IS I S IS α-spectrin SH3 domain (~ C- 13 C restraints) M.H. Levitt, Spin Dynamics Oschkinat et al., Nature (2002) Conventional approaches rely on measurements of through-space 13 C- 13 C & 13 C- 15 N dipolar couplings in 2D/3D correlation spectra

3 Protein Structure by MAS Solid-State NMR D γγ / r 3 IS I S IS α-spectrin SH3 domain (~ C- 13 C restraints) M.H. Levitt, Spin Dynamics Oschkinat et al., Nature (2002) Conventional approaches rely on measurements of through-space 13 C- 13 C & 13 C- 15 N dipolar couplings in 2D/3D correlation spectra

4 Protein Structure by MAS Solid-State NMR HET-s Prion Fibrils Anabaena Sensory Rhodopsin Type-III Secretion System Needle Meier et al. Science (2008) Ladizhansky et al. Nature Methods (2013) Lange et al. Nature (2012) High-resolution structural and dynamic analysis is now possible for proteins up to ~300 aa and multi-mda assemblies of smaller subunits

5 Long-Range Distances Are Most Critical D γγ / r 3 IS I S IS One bottleneck is the collection of large numbers of unambiguous >~5-6 Å distance restraints: small couplings, multispin effects, low S/N

6 Solid-State NMR of Proteins Modified with Paramagnetic Tags γ e / γ 660 H Intentionally introduce paramagnetic centers at specific sites as long-range structural probes due to large e -n couplings

7 Paramagnetic Effects in MAS SSNMR Spectra Paramagnetic shifts (contact & PCS) Paramagnetic relaxation paramagnetic diamagnetic diamagnetic paramagnetic Frequency Frequency Contact shifts: e -density at nucleus, negligible for e -n distances > ~5 Å PCS: centers with large electron g-anisotropy (e.g., Co 2+, lanthanides) Relaxation: centers with small g-anisotropy (e.g., nitroxides, Cu 2+, Mn 2+, Gd 3+ )

8 Nuclear Paramagnetic Relaxation Fluctuation of direction/intensity of dipolar field generated by electron spin at nucleus leads to enhanced nuclear relaxation

9 Nuclear Paramagnetic Relaxation 2 2C 3T1 7 e T1 e 1 µ Γ1 γ β C= nge ess+ ren 1+ ωnt1 e 1+ ωet1 e 15 4π Γ Γ C 4T + 3T + 13T 1e 1e 2 1ρ 6 1e ren 1+ ωnt1 e 1+ ωet1 e Solomon, Phys. Rev. 99 (1955) 559 ( 1)

10 Nuclear Paramagnetic Relaxation PRE effects can be large for nuclei ~20 Å from paramagnetic center Effects can be modulated by using different paramagnetic centers Transverse PRE directly proportional to T 1e (i.e., slowest relaxing centers cause largest PREs) Longitudinal PRE largest when 1/T 1e ~ nuclear Larmor frequency (in angular units)

11 Electronic Relaxation Times Ln 3+ Ru 3+ (5/2) Fe 3+ (5/2) Gd 3+ (7/2) Cu 2+ (1/2) Mn 2+ (5/2) NO (1/2) T 1e (s) Typical T 1e values are in the range to 10-7 s T 1e values approximately the same for proteins in solution and hydrated proteins in solid RT Bertini & Luchinat, Coord. Chem. Rev. (1996)

12 Concept for PRE Based Protein SSNMR Electron-nucleus distances monitored simultaneously via resonance intensities in simplest 2D or 3D SSNMR spectra Sengupta, Nadaud & Jaroniec, Acc. Chem. Res. 2013, 46, 2117

13 Concept for PRE Based Protein SSNMR Electron-nucleus distances monitored simultaneously via resonance intensities in simplest 2D or 3D SSNMR spectra Sengupta, Nadaud & Jaroniec, Acc. Chem. Res. 2013, 46, 2117

14 Concept for PRE Based Protein SSNMR Electron-nucleus distances monitored simultaneously via resonance intensities in simplest 2D or 3D SSNMR spectra Sengupta, Nadaud & Jaroniec, Acc. Chem. Res. 2013, 46, 2117

15 Spin Labeling of Proteins Hubbell, Curr. Opin. Struct. Biol. (1994) Philippe Nadaud R1/R1 side-chains placed at solvent-exposed aa K28 or T53 Protein fold not affected Diluted in microcrystals with unlabeled/diamagnetic protein GB1 (56 aa)

16 Paramagnetic Protein Samples for SSNMR 12 C, 14 N protein, R1 13 C, 15 N protein, R1 3:1 Microdialysis (MPD:isopropanol) Protein microcrystals Pauli et al., JMR (2000) McDermott et al., JBNMR (2000) Martin & Zilm, JMR (2003) Franks et al., JACS (2005)

17 SSNMR of Spin Labeled GB1-T53C Mutant Diamagnetic Spin-Labeled Ac Nadaud, Helmus, Höfer & Jaroniec, J. Am. Chem. Soc. 2007, 129, 7502

18 SSNMR of Spin Labeled GB1-T53C Mutant Diamagnetic Spin-Labeled Ac Nadaud, Helmus, Höfer & Jaroniec, J. Am. Chem. Soc. 2007, 129, 7502

19 SSNMR of Spin Labeled GB1-T53C Mutant Diamagnetic Spin-Labeled T49 G41 T51 T25 N37 D22 G9 V29 V21 Ac I6 W43 A20 E56 Nadaud, Helmus, Höfer & Jaroniec, J. Am. Chem. Soc. 2007, 129, 7502

20 SSNMR of Spin Labeled GB1-T53C Mutant Diamagnetic Spin-Labeled T49 G41 T51 T25 N37 D22 G9 V29 V21 Ac I6 W43 A20 E56 Signals from nuclei within ~10-12 Å of spin label are suppressed by large transverse PRE effects (mainly during initial 1 H- 15 N CP) Nadaud, Helmus, Höfer & Jaroniec, J. Am. Chem. Soc. 2007, 129, 7502

21 Qualitative Long-Range Distance Restraints Philippe Nadaud Nadaud, Helmus, Höfer & Jaroniec, J. Am. Chem. Soc. 2007, 129, 7502

22 Initial SSNMR Studies of 13 C, 15 N-Metalloproteins Pintacuda, Giraud, Pierattelli, Bockmann, Bertini, Emsley, Angew. Chem. Int. Ed. 2007, 46, 1079 Balayssac, Bertini, Lelli, Luchinat, Maletta, JACS 2007, 129, 2218

23 PRE Tuning by Other Paramagnetic Centers Ermacora et al., PNAS (1992) Ebright et al. Biochemistry (1992) Species logk EDTA-M S T 1e (ns) Γ NO 2 /Γ M 2 Zn Cu /2 ~2 ~50 Mn /2 ~10 ~0.85 nitroxide - 1/2 ~100 1 Tune longitudinal and transverse PREs by using paramagnetic centers with different electronic properties

24 Quantitative Restraints via EDTA-Cu 2+ Tags & R 1 PREs Zn 2+ Cu 2+ Cu 2+ Mn 2+ NO Mn 2+ Nadaud et al. JACS 2009, 131, 8108; Nadaud et al. JACS 2010, 132, 9561

25 Determination of Protein Fold Ishita Sengupta Philippe Nadaud

26 15 N Longitudinal PREs for GB1-EDTA-Cu 2+ Mutants Ishita Sengupta Philippe Nadaud > N PREs (4-5 per aa) for set of 6 Cu 2+ /Zn 2+ GB1 mutants in ~2-3 weeks

27 Quantitative Long-Range Distance Restraints Γ = R (Cu ) R (Zn ) N N 2+ N Quantitative 15 N-Cu 2+ distances in ~10-20 Å range accessible

28 Comparison of Experimental & Predicted PREs: Limited Data Set from Initial Studies β4 β1 β2 α β3 r 2 = 0.94 RMSD = 0.04 s -1 Backbone torsion angles fixed to GB1 values Conformation of EDTA-Cu 2+ refined subject to PRE restraints Good agreement for PRE values > ~0.1 s -1 Nadaud, Helmus, Kall & Jaroniec, J. Am. Chem. Soc. 2009, 131, 8108

29 Effect of Intermolecular Electron-Nucleus Interactions on PRE Measurements ~25% ~15% ~10% ~15-20% dilution of 13 C, 15 N-protein in natural abundance matrix appears to be optimal, though not critical; several elevated PREs observed at ~10% dilution (secondary Cu 2+ binding site) Nadaud, Sengupta, Helmus & Jaroniec, J. Biomol. NMR 2011, 51, 293

30 Observation of Cu 2+ Sites by Solution NMR ~25% ~15% ~10% For super-stoichiometric [Cu 2+ ]/[protein] ratios the Cu 2+ ions appear to bind to surface Asp and Glu side-chains Nadaud, Sengupta, Helmus & Jaroniec, J. Biomol. NMR 2011, 51, 293

31 Rapid Acquisition of SSNMR Protein Spectra 2D 15 N- 13 CO-S 3 40 khz MAS GB1-28EDTA-Cu 2+ Sample: 1 mg (~150 nmol) Experiment time: 7 min! Fast data acquisition facilitated by rapid MAS, low-power RF & Cu(II)-tags (same idea as PACC approach introduced before by Ishii and others) Nadaud, Helmus, Sengupta & Jaroniec, J. Am. Chem. Soc. 2010, 132, 9561

32 Rapid 3D SSNMR: Sequential Assignments Complete backbone walk from two 3D SSNMR spectra of ~3 h each Sensitivity can be further improved with deuterated proteins & 1 H detection Nadaud, Helmus, Sengupta & Jaroniec, J. Am. Chem. Soc. 2010, 132, 9561

33 Refinement with X-ray Data and PREs No PREs Torsions for helix & strands fixed to X-ray values, loops randomized Collaboration with C. Schwieters (NIH)

34 Refinement with X-ray Data and PREs No PREs With ~230 PREs Torsions for helix & strands fixed to X-ray values, loops randomized Collaboration with C. Schwieters (NIH)

35 Refinement with TALOS+ and PREs De novo calculation gives correct global fold with 1.8 Å bb RMSD vs. X-ray Sengupta, Nadaud, Helmus, Schwieters & Jaroniec, Nature Chem. 2012, 4, 410

36 Compact High-Affinity Cu 2+ Binding Tags Synthesis based on: Lacerda et al., Polyhedron (2007) TETAC Ishita Sengupta Min Gao Rajith Arachchige

37 PRE Measurements: 28TETAC-Cu 2+ GB1 Sengupta et al. J. Biomol. NMR 2015, 61, 1

38 PRE Measurements: 28TETAC-Cu 2+ GB1 Signals from nuclei within ~10 Å of Cu 2+ center strongly attenuated due to transverse PREs Sengupta et al. J. Biomol. NMR 2015, 61, 1

39 PCS Measurements in Co 2+ Tagged Proteins δ PCS = ( 3cos 2 θ 1) 1 Δχax + π χ θ ϕ 3 12 r 3 2 en 2 Δ rh sin cos Calculated 13 C PCS (ppm) Experimental 13 C PCS (ppm) Zn 2+ Co 2+ Rajith Arachchige

40 Structure determination of a Co 2+ metalloprotein aided by PCS restraints Bertini, Bhaumik, De Paepe, Griffin, Lelli, Lewandowski, Luchinat JACS 2010, 132, 1032

41 Structure determination with PCS restraints from 4MMDPA-Co 2+ proteins and CS-Rosetta Li, Pilla, Yang et al. JACS 2013, 135, MMDPA: Su, Otting et al. JACS 2008, 130, 10486

42 Oligomeric State of Membrane-Bound 7-Helix Sensory Rhodopsin from PREs S26R1 Wang, Munro, Kim, Jung, Brown & Ladizhansky, JACS 2012, 134, 16995

43 SSNMR of Spin Labeled Human PrP Fibrils A113C-Ac-MTSL A113C-MTSL Theint Theint

44 Higher Order huprp Fibril Architecture 0.48 MPL η = 1.99 MW ~5 nm Intermolecular PREs ~5 nm

45 Solvent Interfaces via 15 N PREs with Cu 2+ -EDTA: Similar to earlier work by Ishii, Reif < 0.1 s -1 (protected) s -1 > 0.25 s -1 (exposed) n.d. Darryl Aucoin

46 Summary Paramagnetic tags can be used as unique structural probes in MAS solid-state NMR with many potential applications to biological solids: - Quantitative long-range distance measurements - Protein fold determination - Probing intermolecular contacts - Spectral editing & sensitivity enhancement - Identification of ligand binding sites -

47 Acknowledgments Philippe Nadaud Jonathan Helmus Ishita Sengupta Rajith Arachchige Min Gao Dwaipayan Mukhopadhyay Theint Theint Darryl Aucoin Charles Schwieters (NIH) Witold Surewicz (CWRU)